The accessory proteins of complex retroviruses typically have multiple functions that contribute to pathogenesis in vivo. The Vpx protein of human immunodeficiency virus type 2 (HIV-2) is known to be necessary and sufficient for nuclear import of the viral pre-integration complex and has been recently shown to bind and enhance degradation of the MHC class II associated protein, invariant chain (Ii). The goal of this proposal is to characterize these two distinct functions of Vpx. The first aim of this study is to identify the nuclear localization signal (NLS) of Vpx and define the pathway through which Vpx accesses the nucleus. The NLS will be identified by nuclear localization studies and then used as "bait" in a yeast two-hybrid screen to identify proteins important for Vpx nuclear import. Positive clones will be subsequently confirmed for Vpx-interaction by a battery of assays. The second aim is to determine how Vpx enhances Ii degradation and delineate the effects of Vpx on the class II pathway. MHC class II pathway components, including Ii, will be analyzed in cells infected with Vpx(+) and Vpx(-) viruses. A Vpx-inducible cell line will be developed and used to dissect the mechanisms by which Vpx enhances degradation of Ii and affects the MHC class I pathway. These studies will further the understanding of the role of Vpx in HIV-2 replication and pathogenesis and contribute to the development of HIV inhibitory therapies.